Start-up of continuous butyric acid fermentor

ABSTRACT

A process for starting up the continuous production of butyric acid by fermentation of a medium containing a starting amount of a nutrient substrate and a microorganism that converts the nutrient into butyric acid, that avoids an excess of nutrient in the fermentation broth and comprises the steps of (1) fermenting in a fermentor in batch style an amount of the medium that is substantially less than the working volume of the fermentor until the amount of the nutrient not yet converted is no more than a small fraction of the starting amount, (2) adding medium to the fermentor without removing any of the fermentation broth, until it contains approximately its working volume, then, (3) while continuing the addition, removing the fermentation broth containing the butyric acid product at a continuous rate that maintains a constant volume of medium in the fermentor, and thereafter maintaining continuous operation of the fermentation.

This invention relates to the production of butyric acid by theanaerobic fermentation of carbohydrates.

BACKGROUND OF THE INVENTION

Butyric acid is used in butter and rum flavors, and its ethanol ester(ethyl butyrate) is used in fruit flavors. Other esters of butyric acidare also used in flavors. The production of such natural flavoringredients by fermentation has become a desirable alternative to theirphysical extraction from plants because it avoids the disadvantages ofsuch extraction while retaining the recognized "natural" designation,based on the ruling by the FDA (21 CFR 101.22.a.3) that products offermentation are considered natural provided that the starting substrateis a natural material such as glucose or sucrose.

The production of butyric acid by fermentation is well known. Oftenacetic acid is also produced in these fermentations; acetic acid andseveral of its esters (particularly ethyl acetate) are also of interestto the flavor industry. Usually a member of the genus Clostridium isused for the butyric acid fermentation, and the fermentation isconducted anaerobically. Substrates comprising sugars such as glucose,sucrose, and fructose, or carbohydrates that break down into such sugarsare conventionally used, for example, in batch, semi-batch ("fed-batch")or continuous operations.

Fermentation processes that can produce butyric acid are described, forinstance, in U.S. Pat. Nos. 4,539,293 and 4,814,273, and in an articlepublished in Appl. Microbiol. Biotechnol. (34, 2, 172-77) 1990, by D.Michel Savin, D. R. Marchal, and J. P. Vandecasteele of the DivisionBiotechnoloqie et Environnement, Institut Francais du Petrole, thatdescribes the metabolic behavior and production performance ofClostridium tyrobutyricum in a continuous culture.

U.S. Pat. No. 4,814,273 describes the fermentation in batch mode of alactate salt to butyrate salt using Butyribacterium methylotrophicum (B.methylotrophicum), and following that fermentation with a secondfermentation in the same mixture that converts a carbohydrate to lacticacid using a Lactobaccillus, preferably after removing the cells of themicroorganism used in the first step. Productivity is usually higherwith continuous operation (as suggested by the good results reported byMichel Savin et al. in the above literature publication), becauseunproductive downtime for repeatedly draining the fermentor, refillingthe fermentor, and growing the culture is not required.

For continuous operation, the conventional arrangement is a chemostat,in which nutrient medium is fed to a stirred fermentor tank at acontrolled rate and broth is removed at the same rate, so that the levelin the fermentor remains constant. The dilution rate (expressed asliters.liter⁻¹.hr⁻¹, or simply hr⁻¹) must not exceed the maximum growthrate of the organism, and the formation of the desired product must beat least partially associated with the growth of the microorganism.Complex nutrient supplements (e.g., yeast extract) are conventionallyused, as additives or in complex media compositions, to obtain a highyield of product, as in U.S. Pat. No. 4,814,273, although they may havesome disadvantages; for example, these complex materials, which havevariable compositions, can support growth of a wide variety oforganisms, so that they render the fermentor more susceptible tounpredictable contamination. Furthermore, they usually are notcompletely soluble, which can lead to abrasion problems with heatexchangers, pumps, and fermentor agitator blades, and makes the mediumunsuitable for sterilization by filtration, which is sometimes desirableto reduce energy costs or if the medium contains heat-labile components.

For these reasons a nutrient substrate essentially comprising a compoundhaving a chemically definable structure, such as glucose or sucrose, andall other medium components having a chemically definable structure maybe desirable, although the yield of the desired product may not be ashigh and small amounts of specific additional nutrients may have to beadded.

The published disclosures of continuous fermentation work do not addressthe problems that are encountered in the conventional procedures used tostart up a continuous butyric acid fermentation process. The fermentoris conventionally operated in batch mode until the concentration of themicroorganism is sufficiently high, then addition of the fermentationmedium (feedstock) and removal of the medium containing the butyric acidproduct (fermentation broth) at a continuous rate that maintains aconstant volume of medium in the fermentor is continued. However, whenfeeding of the medium begins, it causes drastic changes in theestablished balance of the ingredients of the fermentation broth; thebutyric and acetic acid concentrations typically decrease and theresidual substrate concentration correspondingly increases. As much asseveral days may be required before the proper concentrations of theproducts in the medium are re-established. In a commercial process thistransitory reduction in butyric acid yield can be very costly, and thepresence of excessive substrate can interfere with downstreampurification of the products.

There is clearly a need for a method of making a smooth transition frombatch to continuous operating mode, without such undesirable changes inthe established balance of the ingredients.

SUMMARY OF THE INVENTION

According to the invention, a process for the production of butyric acidby the fermentation of an aqueous medium containing a starting amount ofa nutrient and a microorganism that converts the nutrient into butyricacid, involves a procedure for starting up the continuous butyric acidfermentor that eliminates this transition period, with no excesssubstrate or decrease in butyric acid concentration.

The process comprises a specific sequence of steps, starting with (1)fermenting in a fermentor an amount of the medium that is substantiallyless than the working volume of the fermentor, which in effect comprisesgrowing the culture in batch mode as described above. Preferably thefermentor is filled only until it contain from 20% up to 60% of itsworking volume; and the fermentation is allowed to proceed in batchmode, without any additions of the nutrient substrate that wouldnormally be made in fed-batch mode.

Step (2) is started when the amount of the nutrient not yet converted isno more than a very small selected fraction of the starting amount,preferably when the nutrient substrate concentration has been depletedto a level below 1 gram per liter while still maintaining the vitalityof the microorganism, and involves adding medium to the fermentorwithout removing any of the fermentation broth, until it containsapproximately its working volume, the rate of addition being not morethan the maximum rate of conversion of the nutrient.

Then in step (3), while continuing the addition, the fermentation brothcontaining the butyric acid product is removed at a continuous rate thatmaintains a constant volume of medium in the fermentor, and thereaftercontinuous operation of the fermentation is maintained.

During step (2), while medium is added to the fermentor without removingany of the fermentation broth, the rate of addition (feedrate) may beset so that the dilution rate is equal to the desired dilution ratebased on that final working volume in continuous mode. Alternatively andpreferably, the dilution rate is set based on the initial volume, andthe feedrate is increased as the level increases ("ramped" feedrate), sothat the dilution rate remains approximately constant for maximumefficiency.

Once the fermentor working volume is reached, the step of removing thereaction broth mixture is started at a rate that maintains the volume ofmedium in the fermentor, whereby continuous operation is established. Asmentioned above, the transition from batch to continuous operationoccurs with no decrease in butyric acid concentration.

The invention can be successfully applied to any fermentation using anutrient medium that is suitable for conventional fermentation that canbe operated continuously, including those sometimes preferred for theirhigh yield and using complex nutrient supplements such as yeast extract,but the following description and examples use nutrient mediaessentially comprising preferred examples of chemically definablecompounds, namely, glucose and sucrose, to eliminate as far as possibleuncontrolled variables and demonstrate the operability of the preferredmethodology or best mode of the invention in the absence of extraneousand conventional factors that are available if desired but are not apart of the invention itself. Other nutrient compounds having definablestructures, such as fructose and glycerol, are nutrients for members ofthe genus Clostridium that may be used in carrying out the invention,such as Clostridium butyricum (C.butyricum), C. tyrobutyricum, C.pasteurianum, C. acetobutyricum, C. beijerinckii, or C.methylotrophicum.

One of the parameters important to the successful implementation of theinvention is the substrate concentration of the feed medium. Achievementof a smooth transition from batch to continuous mode is difficult athigh substrate concentrations, which may make steady state continuousoperation impractical. Preferably, the substrate concentration in thefeed is such that the carbon is present at 14 to 17 g/l, and preferablyat no more than 16 g/l. For example, glucose, which contains 40% carbon,and sucrose, with 42% carbon, give the preferred maximum carbon level of16 g/l at concentrations of 40 g/l and 38 g/l respectively.

In the following examples, which further illustrate the invention,conventional additives including vitamins such as d-biotin,p-aminobenzoic acid (PABA), choline, folic acid, inositol, niacin,pantothenic acid, pyridoxine, riboflavin, and thiamine (most preferably,at least d biotin and PABA), and including minerals such as alkali metaland ammonium phosphates and sulfates of metals such as copper, magnesiumor iron, are preferably used in the fermentation medium, to demonstratethe applicability of the invention to preferred conventionalfermentation environments. In addition, the pH was conventionallymaintained at about 6.2 by adding alkali metal hydroxide.

EXAMPLE 1 Start-up With a Glucose Medium at a Constant Feedrate

A 2.4 liter fermentor containing water was sterilized in place at 121°C. for 30 minutes, then the water was removed aseptically. One liter ofthe medium shown in Table 1 (except the ferrous sulfate) was preparedand transferred to the fermentor through a 0.2-micron sterilizing filterwhich had been autoclaved at 121° C. The ferrous sulfate was preparedseparately as a concentrated solution, acidified to pH 2.5 by additionof citric acid, then transferred through a 0.2-micron sterilizing filter(which also had been autoclaved at 121° C.) into the fermentor. About0.35 g/l of sterile antifoam was also added.

The stirring speed and temperature were maintained at 700 rpm and 35° C.respectively. Nitrogen was sparged throughout the fermentation at a rateof 0.1 standard liters per liter per minute. The fermentor wasinoculated with 35 ml of a 12-hour culture of Clostridium butyricum ATCC860 (American Type Culture Collection, Rockville, Md.). The pH wasmaintained at 6.2 by automatic addition of aqueous (450 g/l) potassiumhydroxide.

After about 12 hours glucose was nearly depleted, the butyric acidconcentration was 13 g/l, and the acetic acid concentration was 10 g/l.At that time feed of the medium shown in Table 1 (except ferroussulfate) through a 0.2-micron sterilizing filter was begun at a feedrateof 180 ml/hr. As explained above, the ferrous sulfate was fed separatelyas a concentrated solution which had been acidified to pH 2.5 withcitric acid. Sterile antifoam was also fed at a rate equal toapproximately 0.35 grams per liter of medium feed. This fed-batchoperation continued until the fermentor volume was 1.8 liters. At thattime effluent removal began, and steady-state volume and dilution ratewere established at 1.3 liters and 0.1 hr⁻¹ respectively. No appearanceof residual glucose was observed, the butyric acid concentration did notdrop below 13 g/l, and the acetic acid concentration did not drop below10 g/l any time after the completion of the batch phase.

                  TABLE 1                                                         ______________________________________                                        Glucose-based Fermentation Medium                                             Component       Concentration, g/l                                            ______________________________________                                        Glucose         38.5                                                          Ammonium acetate                                                                              5.0                                                           KH.sub.2 PO.sub.4                                                                             0.5                                                           K.sub.2 HPO.sub.4                                                                              0.38                                                         MgSO.sub.4.7H.sub.2 O                                                                         0.2                                                           FeSO.sub.4.7H.sub.2 O                                                                         0.1                                                           NH.sub.3 H.sub.2 PO.sub.4                                                                      1.36                                                         p-aminobenzoic acid*                                                                           0.01                                                         d-biotin         0.001                                                        Softened water  balance                                                       ______________________________________                                         *Potassium salt.                                                         

EXAMPLE 2 Start-up With a Sucrose Medium at a Constant Feedrate

The fermentor was prepared in exactly the same manner as in Example 1except for the following:

the medium shown in Table 2 was used.

the magnesium sulfate and ferrous sulfate were prepared together as aconcentrate, acidified to pH 2.5 with citric acid, then added to thefermentor separately from the rest of the medium.

the magnesium sulfate/ferrous sulfate concentrate was sterilized byautoclaving rather than by filtration.

pH was controlled with 500 g/l NaOH instead of 450 g/l KOH.

The fermentor was inoculated with 35 ml of a 12-hour culture ofClostridium butyricum ATCC 860. After about 12 hours sucrose was nearlydepleted, the butyric acid concentration was 12 g/l, and the acetic acidconcentration was 5 g/l. At that time feed of the medium shown in Table2 was begun at a feedrate of 180 ml/hr. As explained above, the ferroussulfate and magnesium sulfate were fed separately as a concentratedsolution which had been acidified to pH 2.5 with citric acid. Sterileantifoam was also fed at a rate equal to approximately 0.4 grams perliter of fermentation medium. This fed-batch operation continued untilthe fermentor volume was 1.8 liters. At that time effluent removalbegan, and steady state volume and dilution rate were established at 1.3liters and 0.1 hr⁻¹ respectively. No appearance of residual sucrose wasobserved, the butyric acid concentration did not drop below 12 g/l, andthe acetic acid concentration did not drop below 5 g/l any time afterthe completion of the batch phase.

                  TABLE 2                                                         ______________________________________                                        Sucrose-based fermentation medium                                             Component       Concentration, g/l                                            ______________________________________                                        Sucrose         36.7                                                          Ammonium sulfate                                                                               4.28                                                         KH.sub.2 PO.sub.4                                                                             0.5                                                           K.sub.2 HPO.sub.4                                                                              0.38                                                         MgSO.sub.4.7H.sub.2 O                                                                         0.1                                                           FeSO.sub.4.7H.sub.2 O                                                                         0.1                                                           p-aminobenzoic acid*                                                                           0.01                                                         d-biotin         0.001                                                        Softened water  balance                                                       ______________________________________                                         *Potassium salt.                                                         

EXAMPLE 3 Start-up With a Sucrose Medium and the Feedrate Increased("Ramped") With Increasing Fermentor Volume

The fermentor was prepared and inoculated exactly as described inExample 2. Sucrose was nearly depleted after 12 hours, and the butyricand acetic acid concentrations were 12 and 5 g/l respectively. Mediumfeed was started at a rate of 100 ml/hr, i.e., at a dilution rate of 0.1hr-1. This dilution rate was maintained approximately constant duringthe fed-batch phase by increasing the feedrate hourly based on currentvolume. For example, after one hour the volume was approximately 1.1liters and the feedrate was increased to 110 ml/hr. The fermentor wasoperated in this manner until the volume was 1.8 liters, then brothremoval began and the steady-state operating volume and dilution rate of1.3 liters and 0.1 hr³¹ 1 respectively were established. No appearanceof residual sucrose occurred, butyric acid concentration did not dropbelow 12 g/l, and acetic acid concentration did not drop below 5 g/l atany time after the completion of the batch phase.

I claim:
 1. A process for the production of butyric acid by thefermentation of an aqueous medium containing a starting amount of amicroorganism from the genus Clostridium and a nutrient for themicroorganism consisting of a nutrient selected from the groupconsisting of fructose, glucose, glycerol and sucrose, that themicroorganism is converting into butyric acid, comprising the steps of(1) fermenting in a fermentor an amount of the medium that issubstantially less than the working volume of the fermentor until theamount of the nutrient not yet converted is no more than a smallfraction of the starting amount, (2) adding medium to the fermentorwithout removing any of the fermentation broth, until it containsapproximately its working volume, then, (3) while continuing theaddition, removing the fermentation broth containing the butyric acidproduct at a continuous rate that maintains a constant volume of mediumin the fermentor, and thereafter maintaining continuous operation of thefermentation; wherein the medium contains no more than 17 grams ofcarbon in the structure of the nutrient substrate per liter of themedium.
 2. A process for the production of butyric acid as claimed inclaim 1, in which during the step (2) of adding medium to the fermentorwithout removing any of the fermentation broth, the rate of addition isequal to the desired dilution rate based on the final working volume,which rate is not more than the maximum rate of conversion of thenutrient.
 3. A process for the production of butyric acid as claimed inclaim 1, in which during the step (2) of adding medium to the fermentorwithout removing any of the fermentation broth, the starting rate ofaddition is based on the initial volume in the fermentor, and isincreased as the level increases, so that the dilution rate ismaintained approximately constant.
 4. A process for the production ofbutyric acid as claimed in claim 1, in which in step (1) the amount ofthe medium that is substantially less than the working volume of thefermentor is from 20% to 60% of the working volume of the fermentor. 5.A process for the production of butyric acid as claimed in claim 1, inwhich at the end of step (1) the amount of the nutrient not yetconverted is less than 1 gram per liter while still sufficient tomaintain the vitality of the microorganism.
 6. The method claimed inclaim 1 in which the nutrient substrate is selected from the groupconsisting of glucose or sucrose.
 7. The method claimed in claim 1 inwhich the medium contains no more than 16 grams of carbon in thestructure of the nutrient substrate per liter of the medium.